ABSTRACT
Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR)in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma (PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Westem blotting,respectively.The SGC7901/VCR cells were treated with 0.1 mg/L vincristine(VCR)alone or in combination with 1,5,10 μmol/L troglitazone for 24 h.PPARγ was measured by electrophoretic mobility shift assay(EMSA).The intracellular concentration of Rhodamine123(Rh123,a fluorescent P-gp substrate)was assayed to evaluate the activity of P-gp.The cell cycle and apoptosis were measured by flow cytometry.The results showed that the P-gp was increasingly expressed in SGC7901,BGC823 and SGC7901/VCR cells in turn,suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp.In the SGC7901/VCR cells,the expression level of total PPARγ was increased,however,the protein level and activity of PPARγ,in the nuclei of cells decreased significantly.Troglitazone elevated the PPARγ,activity in SGC7901/VCR cells in a dose-dependent manner.Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner.We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner.Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner.It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity.Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells.It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.
ABSTRACT
The relationship between intracelluar trypsinogen activation and NF-r,B activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist (carbachoi) hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active pro- tease inhibitor (pefabloc) and NF-кB inhibitor (PDTC) in vitro. Intracelluar trypsin activity was measured by using a fluorogenie substrate. The activity of NF-кB was monitored by using electro- phoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-кB in pancreatic acinar cells treated with high concertrations of carbachol (10-3 mol/L) in vitro was significantly decreased as compared with control group (P＜0.01). The addi- tion of 10-2mol/L PDTC resulted in a significant decrease of NF-кB activities in pancreatic acinar cells after treated with high concertrations of carbachol (10-3 mol/L) in vitro, but the intracelluar trypsinogen activity was not obviously inhibited (P＞0.05). It was concluded that intracelluar trypsi- nogen activation is likely involved in the regulation of high concertrations of carbachol-induced NF-кB activation in pancreatic acinar cells in vitro. NF-кB activation is likely not necessary for high concertrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.